PROJECT SUMMARY Profiling of Extracellular Vesicles (EV) has emerged as a highly promising field for discovery of tumor-relevant biomarkers from biofluids, such as blood, urine, CSF or others. The use of biofluid EVs offers numerous advantages in clinical setting, including non-invasive collection, a suitable sample source for longitudinal disease monitoring, better screenshot of tumor heterogeneity, higher stability and sample volumes, faster processing times, lower rejection rates and costs compared to their tissue counterpart. Despite the immense potential, the data on EV phosphoproteomes are virtually non-existent. In this NIH SBIR Fast-Track study, we will further develop a novel urine analysis platform based on EVtrap beads for complete capture of extracellular vesicles and in-house developed proteome extraction and analysis approach into highly efficient and reproducible method for discovery and detection of cancer biomarkers. We have already utilized this approach to generate a new panel of protein/phosphoprotein markers from urinary EVs for sensitive and non-invasive detection and monitoring of bladder cancer. During this project, the discovered biomarkers panel will be validated and refined to demonstrate clinical utility. The following aims will be completed in the Phase I of the proposal: Aim #1: Develop and optimize EVtrap for large-scale and high-throughput EV isolation. Aim #2: Determine the feasibility of the current bladder cancer biomarkers and refine the panel. The following aims will be completed in the Phase II of the proposal: Aim #1: Adapt EVtrap to an existing automated device for high-throughput EV capture and cargo analysis. Aim #2: Validate and standardize the final panel for clinical utility. By the completion of this project, a biomarker discovery platform from urine will be developed, and bladder cancer monitoring assay will be validated that can overcome the limitations of current approaches, and thus could have an enormous public health impact and market potential.